Reduction of milk glycans and their degradation products in the neonate gut

ABSTRACT

Methods of reducing milk glycans, and thus enteric pathogens that use such glycans as carbon sources, in the gut of nursing mammals.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 16/420,032, filed May 22, 2019, which is a continuation of U.S.patent application Ser. No. 15/533,575, filed Jun. 6, 2017, now U.S.Pat. No. 10,350,249, issued Jul. 16, 2019, which is a US National Phaseof International Application No. PCT/US2015/065323, filed Dec. 11, 2015,which claims the benefit of priority to U.S. Provisional PatentApplication No. 62/091,298, filed Dec. 12, 2014, which are incorporatedby reference for all purposes.

REFERENCE TO SUBMISSION OF A SEQUENCE LISTING AS A TEXT FILE

The Sequence Listing written in file SequenceListing 1206243.txt createdon Aug. 17, 2020, 922 bytes, machine format IBM-PC, MS-Windows operatingsystem, is hereby incorporated by reference in its entirety for allpurposes.

BACKGROUND OF THE INVENTION

Sialic acid, released from milk glycans, has been identified as aprimary carbon source driving increases in populations ofEnterobacteriaceae and Clostridiaceae, which leads to diarrhea innursing mammals. Data has demonstrated that the bacterial sialidasesliberate sialic acid which is consumed by populations ofEnterobacteriaceae in the gastrointestinal tract of nursing pigs. Thissialic acid is released from milk glycans and host glycans by abacterial enzyme, sialidase also known as neuraminidase (EC 3.2.1.18).The present technology demonstrates the reduction in sialic acid andN-acetylglucosamine, by delivery of an organism which consumes sialicacid, sialic acid-containing milk glycans, N-acetylglucosamine, orN-acetylglucosamine-containing milk glycans to limit availableconcentrations of these glycans in vivo. Specifically, this research hasshown that this reduction is effective in preventing colonization bythese pathogens in nursing animals through this mechanism. Evidence forreduction of Enterobacteriaceae by sialic acid consuming lactobacillusis presented.

There are currently no methods which selectively prevent or reduce thegrowth of Enterobacteriaceae populations in the gut of animals byreducing their nutritional niche in the gut. Piglet diarrhea (scour) andpopulations of Enterobacteriaceae and Clostridiaceae are controlled atpresent by antibiotics or vaccines, which do not selectively inhibit thepopulations of Enterobacteriaceae and prevention strategies.

BRIEF SUMMARY OF THE INVENTION

This disclosure pertains generally to reducing free milk glycans andmore specifically to reducing free milk glycan monomers generated by theneonate gut microbiota to eliminate colonization by dysbiotic microbiomemembers.

From the description herein, it will be appreciated that that thepresent disclosure encompasses multiple embodiments which include, butare not limited to, the following:

1. A method of altering the composition of the gut bacterial microbiomein nursing non-human mammals to prevent and treat disease, the methodcomprising:

administering to a nursing mammal a dose of an agent that limits theavailability of at least one of milk glycans and milk glycan monomers inthe nursing mammal's gut;wherein the limited availability of said at least one of milk glycansand milk glycan monomers reduces total populations of enteric pathogensand related organisms in the nursing mammal's gut.

2. The method of embodiment 1, wherein said agent comprises a microbethat competitively utilizes at least one of milk glycans and milk glycanmonomers.

3. The method of embodiment 1, wherein said agent comprises amicroorganism with the ability to consume a milk glycan monomer.

4. The method of embodiment 1, wherein said agent comprises a bacteriumfrom the genus of at least one of Lactobacillus and Bifidobacterium.

5. The method of embodiment 4, wherein said agent is administered atdose of 10⁴ to 10¹² colony forming units (CFU).

6. The method of any of embodiments 3-5, further comprisingadministering at least one prebiotic agent that stimulates colonizationof the microorganism in the mammal's gut.

7. The method of embodiment 1, wherein said agent comprisesBifidobacterium longum subsp. infantis, B. longum subsp. longum, B.breve, or B. pseudocatenulatum, or B. bifidum.

8. The method of embodiment 1:

wherein said agent comprises a species of bacterium from the genus of atleast one of Lactobacillus and Bifidobacterium;

wherein said species are selected for growth on at least one of milkglycans and milk glycan monomers; and

wherein said growth limits availability of said at least one of milkglycans and milk glycan monomers.

9. The method of embodiment 1, wherein said agent comprises at least10¹⁰ colony forming units (CFU) of Lactobacillus reuteri, selected forgrowth on gluconate.

10. The method of embodiment 1, wherein said agent is a microorganismpossessing any combination of the following characteristics:

genes encoding a neuraminidate lyase (nanA) [sialic acid consumption],sialic acid transporter and/or permease, N-acetylglucosamine transporteror permease, N-acetylglucosamine deacetylase (nagA), glucosaminedeaminase (nagB) [nagA and nagB encode for N-acetylglucosamineconsumption and sialic acid consumption], fucose transporter and/orpermease, fucose isomerase, gluconate transporter and/or permease,gluconate kinase, permeases or transporters capable of transportingcomplex glycans composed of one or more of the following monomers;sialic acid, fucose, N-acetylglucosamine; or permeases or transporterscapable of transporting milk glycans.

11. The method of any of embodiments 1-10, wherein said nursing mammalis a species capable of producing milk containing glycans comprised ofat least one of the milk glycan monomers sialic acid, fucose,N-acetylglucosamine, N-acetylgalactosamine, galactose, and glucose.

12. The method of any of embodiments 1-10, wherein said milk glycanmonomers are comprised of at least one of the milk glycan monomersselected from the group consisting of sialic acid, fucose,N-acetylglucosamine, N-acetylgalactosamine, galactose, glucose,gluconate, N-acetylmannosamine, N-acetylmannosamine-6-Phosphate,fuculose-1-phosphate, lactaldehyde, and 1,2-propanediol,galactose-1-phosphate, and galactitol.

13. The method of embodiment 4, wherein said agent is administeredorally in a live form.

14. The method of embodiment 4, wherein said agent further comprises aprebiotic glycan substrate and wherein said prebiotic glycan substrateis utilized by said bacterium.

15. The method of any of embodiments 1-14, wherein said agent isadministered orally in a lyophilized form.

16. The method of any of embodiments 1-15, wherein the mammal is treatedwith antibiotics.

17. The method of any of embodiments 1-15, wherein the mammal is nottreated with antibiotics.

18. The method of any of embodiments 1-17, wherein the mammal is aporcine or bovine mammal.

19. The method of any of embodiments 1-18, wherein the mammal hasdiarrhea.

20. The method of any of embodiments 1-18, wherein the mammal does nothave diarrhea.

21. The method of any of embodiments 1-20, wherein the mammal has anelevated gut concentration of milk glycan monomers or proteobacteria(e.g., Enterobacteriaceae or Bacteroidaceae) prior to the administering

22. The method of embodiment 21, wherein the level of sialic acid in themammal gut (as measured in the mammal's feces) is at least 1 mg per mgtotal protein or the level of fucose is at least 1 mg/g feces, or thelevel of N-acetylglucosamine or N-acetylgalactosamine is at least 10micrograms/g feces prior to the administering.

23. The method of embodiment 21, wherein (i) at least 10% of the totalbacteria community in the mammal's gut is, or (ii) at least 10⁷ cellsper gram gut organ contents are Enterobacteriaceae or Bacteroidaceaeprior to the administering.

24. The method of embodiment 21, wherein the mammal is tested for thepresence of elevated levels of milk glycan monomers or proteobacteriaprior or after the administering.

Further aspects of the technology described herein will be brought outin the following portions of the specification, wherein the detaileddescription is for the purpose of fully disclosing preferred embodimentsof the technology without placing limitations thereon.

BRIEF DESCRIPTION OF THE DRAWINGS

The technology described herein will be more fully understood byreference to the following drawings which are for illustrative purposesonly:

FIG. 1: Graph showing typical piglet E. coli isolate on pig milk sugarconstituent sugars vs. conjugated glycans.

FIG. 2: stacked bar chart showing relative abundance of populations ofBacteroidaceae (in yellow), and in blue, the Enterobacteriaceae arestrongly correlated (r2=0.661, p<0.001) in the feces of young pigs.Communities in weaned animals are boxed.

FIG. 3A: Chart showing taxonomic identity of metagenomic reads annotatedas sialidase enzyme.

FIG. 3B: Chart showing significant sialidase relative abundancedifferences between milk and weaning diets.

FIG. 4: Chart showing free sialic acid concentration in the feces ofnursing and weaned piglets.

FIG. 5: Chart showing average Enterobacteriaceae populations over timein pigs (left axis, bars, nursing, blue; weaned, red), are significantlydifferent (p<0.001) between diets as well as concentrations of freesialic acid, p<0.001 (Right axis, whiskers, nursing, blue; weaned, red).

FIG. 6: Chart showing biogeographical relative abundances ofBacteroidales and Enterobacteriaes in the gut of 14 day old nursingpigs.

FIG. 7: Graph showing treating 14d old pigs by gavage with LactobacillusUCD14261 led to significant reductions in Enterobacteriaceaepopulations.

FIG. 8: Chart showing distinct differences in “At risk” orhigh-Enterobacteriaceae versus “NR” “No risk” pigs prior to gavage withLactobacillus.

FIG. 9: Chart showing “at risk” (AR) or high-Enterobacteriaceae pigscould be rescued by gavage with Lactobacillus to resemble No Risk or NonResponder animals. Letters denote significance groups (a, b; p<0.05).

FIG. 10: Model for PMO Consumption.

DETAILED DESCRIPTION OF THE INVENTION

Any nursing non-human mammal (e.g., a porcine or bovine nursing mammal)can be treated with an agent that reduces the availability of at leastone milk glycan and/or milk glycan monomer (e.g., sialic acid) in thenursing mammal's gut. The reduction of the milk glycan and/or milkglycan monomer will reduce the total population of enteric pathogens inthe mammal's gut by reducing a carbon source of the enteric pathogens.

Exemplary agents that reduce milk glycans and/or milk glycan monomersinclude but are not limited to microorganisms that consume such glycans,for example, Lactobacillus or Bifidobacterium bacteria. Bifidobacteriathat can be included in the compositions administered to the mammalinclude, but are not limited to, B. longum subsp. infantis, B. longumsubsp. longum, B. breve, and B. adolescentis. The Bifidobacterium usedwill depend in part on the target mammal. Lactobacillus that can beincluded in the compositions administered to the mammal include, but arenot limited to, L. acidophilus, L. brevis, L. buchneri, L. casei, L.curvatus, L. delbrueckii, L. fermentum, L. helveticus, L. plantarum, L.reuteri, L. sakei, or L. salivarius or as listed in Table 1 below. Insome embodiments, the Lactobacilli are from the species L. delbrueckii.In some embodiments, the Lactobacilli is Lactobacilli delbrueckiibulgaris, Lactobacilli delbrueckii lactis, Lactobacilli delbrueckiidelbrueckii, or Lactobacilli delbrueckii indicus.

Exemplary bifidobacteria or lactobacilli dosages for formulations caninclude, but are not limited to, 10⁴ to 10¹² colony forming units (CFU)per dose. A further advantageous range is 10⁶ to 10¹⁰ CFU.

In some embodiments, the mammals are further administered a prebioticcompound that increases colonization by the bifidobacteria or thelactobacilli (i.e., the probiotic). The prebiotic can be administeredwith the bifidobacteria or the lactobacilli or can be administeredwithin 12 or 24 hours before or after the probiotic is administered. Forexample, in some embodiments, one or more oligosaccharides that occur inmaternal milk can be administered as the prebiotic. Exemplaryoligosaccharides are described in, e.g., PCT/US2011/043644.Alternatively, galacto-oligosaccharides (GOS) can be administered as aprebiotic. Optionally, the GOS can be enriched for degree ofpolymerization that most benefits the probiotic. See, e.g.,US2014/0037785.

The probiotic (and optionally prebiotic) can be introduced into themammal's diet, for example, as an ingredient of animal feed or added towater or milk.

In some embodiments, the enteric pathogenic bacteria population in themammal's gut prior to administration of the agent includes a populationof Enterobacteriaceae (for example, in some embodiments, E. coli) or theBacteroidaceae (for example, in some embodiments, Bacteroides). In someembodiments, prior to administration the enteric pathogenic bacteriapopulation (e.g., Enterobacteriaceae (for example, in some embodiments,E. coli) or the Bacteroidaceae (for example, in some embodiments,Bacteroides)) is at least, e.g., at least 10% of the total bacterialcommunity, more preferably at least 25% or 50% of the total bacterialcommunity, or preferably at least more than 10′ cells per gram gut organcontents more preferably at least 10¹⁰ cells per gram gut organcontents. In some embodiments, the enteric pathogenic bacteriapopulation reduced in the mammal's gut includes a reduction in apopulation of Enterobacteriaceae (for example, in some embodiments, E.coli) and the Bacteroidaceae (for example, in some embodiments,Bacteroides). In some embodiments, the administration of the agent thatreduces the milk glycan or milk glycan monomer results in a reduction ofthe enteric pathogenic bacteria population (e.g., E. coli population) tobelow, e.g., 50% of the total bacterial community, more preferably lessthan 10% of the total bacterial community, or preferably less than 10¹⁰cells per gram gut organ contents more preferably less than 10′ cellsper gram gut organ contents.

In some embodiments, prior to administration of the agent the level ofsialic acid in the mammal gut (as measured in the mammal's feces) is atleast 1 mg per mg total protein, or in some embodiments, at least 100mg/mg total protein, or the level of fucose is at least 1 mg/g feces, orin some embodiments, at least 10 mg/g feces, or the level ofN-acetylglucosamine or N-acetylgalactosamine is at least 10 micrograms/gfeces or more preferably at least 100 micrograms/g feces. In someembodiments, the administration of the agent that reduces the milkglycan or milk glycan monomer results in a reduction of sialic acid (asmeasured in the mammal's feces) to less than 100 mg/mg total proteinpreferably less than 1 mg per mg total protein, or fucose to less than10 mg/g feces or more preferably less than 1 mg/g feces; orN-acetylglucosamine or N-acetylgalactosamine to less than 100micrograms/g feces or more preferably less than 10 micrograms/g feces.

Exemplary mammals to which the compositions can be administered includeany nursing non-human mammal. Exemplary mammals include, e.g., primates(e.g., monkeys), bovine (e.g., cattle or dairy cows), porcine (e.g.,hogs or pigs), ovine (e.g., goats or sheep), equine (e.g., horses),canine (e.g., dogs), feline (e.g., house cats), camels, deer, donkeys,buffalos, antelopes, rabbits, and rodents (e.g., guinea pigs, squirrels,rats, mice, gerbils, and hamsters). In some embodiments, the mammal canbe under antibiotic treatment (e.g., having received antibiotics withinseven, three or one day of receiving the agent that reduces the milkglycan or milk glycan monomer). In other embodiments, the mammal has notreceived antibiotic treatment or has not received antibiotic treatmentin at least one three or seven days prior to receiving the agent thatreduces the milk glycan or milk glycan monomer.

In some embodiments, the mammals will be showing signs of entericpathogen infection or collibacillosis, e.g., diarrhea, within 1, 2, 3,4, or 5 days prior to treatment with the agent that reduces the milkglycan or milk glycan monomer. Alternatively, the mammal need not haveshown a sign of infection by enteric pathogens and is treatedprophylactically.

Examples

Free milk glycan monomers drive pathogen expansion in nursing pigs. Pigscour or collibacillosis is a major cause of illness and death innursing or weaning pigs worldwide, costing the US alone an estimated$236 million annually through death and disease. Losses resulting fromdelayed growth are likely to greatly exceed this. The disease itself ischaracterized by diarrhea that leads to dehydration and death amonguntreated animals and spreads rapidly within a facility. Costlymitigation procedures must be rapidly implemented and preventativestrategies are clearly more economically viable to swine producers.Preventative strategies currently include vaccination, stringentsanitation procedures, and in-feed antibiotics for weaning animals.Despite these preventative strategies, scour remains a major economiccost to swine producers, and a variety of strategies have been exploredto control its incidence and spread.

The disease itself is caused by any number of Escherichia coli orClostridia strains able to adhere to the gut epithelium and produce anyof a variety of enterotoxins, which leads to tissue damage and diarrheain the animal. In nursing pigs, particularly, this is a deadly infectionand can rapidly lead to severe symptoms or death. While many animals arecolonized by benign populations of E. coli, enterotoxigenic strains ofthe species are able to share the same ecological niche. Further,toxigenic strains can only be distinguished from non-toxigenic strainsby PCR or ELISA-based assays to identify the presence of toxin encodinggenes or the toxins themselves.

The nursing pig's diet is primarily composed of milk from the sow. Pigmilk contains glycans produced by the lactating sow to nourish nursingpigs and these glycans are composed of monomers of glucose, galactose,N-acetylglucosamine, and sialic acid, with a lesser percentage of fucosemonomers (Tao et al 2010). As monomers, these substrates are known to beconsumed by Escherichia and other organisms that are potentiallypathogenic. Other pathogenic organisms may include members of theSalmonella, or the Clostridia, which are also associated withgastrointestinal disease in humans and animals, but E. coli is thepredominant cause of scour in pigs. However, in the form of milkglycans, these complex sugars are indigestible to these organisms. Forexample, a strain of Escherichia coli isolated from the feces of nursingpigs by the inventors is unable to grow on a typical pig milk glycan,sialyllactose, but is able to consume the constituent components (sialicacid, N-acetylglucosamine and lactose) (FIG. 1).

Example Embodiments

Data leading to the present technology. To understand the relationshipof the gut microbiota with pig milk glycans, the inventors completed anexperiment monitoring the temporal changes in the fecal microbiota ofpigs from birth through weaning. Fecal microbial populations remainedstable while the animals were nursing, but changed dramatically atweaning, when milk glycans were removed from the diet. The dominanttaxonomic changes were found during this transition were in the familiesEnterobacteriaceae (which includes E. coli) and the Bacteroidaceae(which includes a genus common to the gut microbiota, Bacteroides) (FIG.2). FIG. 2 shows a stacked bar chart showing relative abundance ofpopulations of Bacteroidaceae (in yellow), and in blue, theEnterobacteriaceae are strongly correlated (r²=0.661, p<0.001) in thefeces of young pigs. Communities in weaned animals are boxed.

Published Bacteroides genomes contain sequences encoding sialidaseenzymes, which may separate the sialic acid moiety from sialyllactose,and create an opportunity for E. coli to thrive in the gut of thenursing animal, where it may not be able to thrive without the activityof this enzyme. Similarly, the activity of beta hexosaminidases, whichremove N-acetylglucosamine monomers from complex glycans also generate aniche for E. coli in this manner, as piglet-isolated E. coli were foundby the inventors to also consume N-acetylglucosamine (FIG. 1). Toconfirm the presence of these enzymes in the animals, genomic microbialDNA was subjected to metagenomic sequencing, to determine theecosystem's total metabolic capabilities, and assign taxonomicidentities to key metabolic roles. Specifically, we sought todemonstrate that the release of sialic acid and N-acetylglucosamine frompig milk glycans is driven by populations of the gut microbiota.

Genes encoding sialidases and beta hexosaminidases were found to belongto members of the gut microbiota. The taxonomic identity of the bacteriahousing these specific enzymes were found to be mostly Bacteroidesassociated with the nursing pigs which diminished when the pigs wereweaned (FIG. 3A). Further, the overall abundance of sequencing readsthat could be mapped to sialidases declined when the diet of the animalschanged to one which contained less of these sugars, suggesting thatthis enzyme is functionally relevant to populations associated with thepig milk diet but not with the weaned diet composed primarily of oats(FIG. 3B).

Reads that could be classified as a sialidase enzyme and identifiedtaxonomically within the Bacteroides were assembled using velvet, tocreate a full-length hypothetical sialidase sequence. One of the contigsfrom this assembly was found to contain a full length sialidase-encodinggene belonging to Bacteroides fragilis, and matched this gene sequenceat 99% nucleotide identity, and was used to generate primers that wouldamplify this sequence from the total fecal DNA sample.

PCR amplification of the gene, using primers matching the hypotheticalsialidase were constructed. These primers successfully amplified asequence from the total fecal DNA, which was subsequently sequenced atthe UC Davis DNA Sequencing core facility. The verified sequence matchedthe hypothetical sequence generated from metagenomic reads at 100%.

In parallel, a representative Bacteroides strain was isolated from fecalsamples of nursing pigs by isolation on Bacteroides Bile Esculin agar, aselective and discriminative medium for the isolation of Bacteroides.Isolated Bacteroides strains were found to contain the sialidase by PCR,using the same primers designed previously, and verified by subsequentDNA sequencing at the UC Davis DNA Sequencing Core. The growth ofBacteroides on sialyllactose was observed, as this organism clearlypossesses a functional sialidase enzyme (data not shown).

Further, sialic acid concentrations in these fecal samples could becompared between nursing and weaning diets and was found to besignificantly greater in samples with greater Bacteroides (and thussialidase enzyme abundance) abundance (FIG. 4). FIG. 5 shows this datafrom another perspective. On days where there is a high relativeabundance of Enterobacteriaceae, there is a high sialic acidconcentration in the feces. On days with low Enterobacteriaceae, thereis a low concentration of sialic acid. FIG. 5 shows AverageEnterobacteriaceae populations over time in pigs (left axis, bars,nursing, blue; weaned, red), are significantly different (p<0.001)between diets as well as concentrations of free sialic acid, p<0.001(Right axis, whiskers, nursing, blue; weaned, red). FIG. 6 shows thatthis effect appears mostly confined to the caecum and colon of thepiglet. There are high Bacteroides in the large intestine but and equalbloom of Enterobacteriaceae in the ensuing feces, suggesting thatBacteroides is indeed creating a substrate (i.e. sialic acid and more)for Enterobacteriaceae to consume. FIG. 6 shows biogeographical relativeabundances of Bacteroidales and Enterobacteriaes in the gut of 14 dayold nursing pigs.

Thus, our data can be summarized as (a) that populations ofEnterobacteriaceae in the gut of nursing pigs was found to correlatewith the abundance of Bacteroides (r2=0.661, p<0.001), (b) and thatthese populations of Enterobacteriaceae cannot, by themselves, consumesialylated pig milk oligosaccharides, but (c) Bacteroides possessenzymes capable of releasing sialic acid from pig milk oligosaccharides,which is (d) associated with increased abundances of sialic acid infeces, which (e) these Enterobacteriaceae can consume.

The synthesis of this knowledge is that monosaccharides released frompig milk glycans leads to increased populations of Enterobacteriaceae inthe gut of nursing pigs, creating an environment where the etiologicalagents of scour can thrive. Specifically, the invention is the knowledgethat by reducing the abundance of mono-, di-, or oligomeric sugars,which may include glucose, galactose, N-acetylglucosamine, sialic acid,or fucose derived from milk glycans, will reduce populations ofEnterobacteriaceae and other potentially pathogenic organisms capable ofconsuming these glycans, their breakdown products, or monosaccharidesand scour will be prevented or reduced in severity.

This could be accomplished by any approach which reduces concentrationsof these monomers or glycans composed of these monomers in the gut. Forexample, introducing a probiotic microorganism which constitutively andcompetitively consumes these freed components or glycans could beintroduced. As an example, and in no way limiting, Table 1 showsexamples of known milk sugar consumers. The species listed have a member(of any subspecies) that possesses genes encoding, for example, aneuraminidate lyase (nanA) [for sialic acid consumption], sialic acidtransporter and/or permease, N-acetylglucosamine transporter orpermease, N-acetylglucosamine deacetylase (nagA), glucosamine deaminase(nagB) [nagA and nagB encode for N-acetylglucosamine consumption andsialic acid consumption], fucose transporter and/or permease, fucoseisomerase, gluconate transporter and/or permease, gluconate kinase,permeases or transporters capable of transporting complex glycanscomposed of one or more of the following monomers; sialic acid, fucose,N-acetylglucosamine; or permeases or transporters capable oftransporting milk glycans.

TABLE 1 Predicted Growth Phenotypes, By Genome Annotations SialicN-acetyl- Species Fucose Acid Gluconate glucosamine LactoseLactobacillus X X X X acidophilus L. amylovorus X X X X L. brevis X X XX X L. buchneri X X X X X L. casei X X X X L. crispatus X X X L.delbrueckii X X L. fermentum X X X L. gasseri X X L. helveticus X X X XL. johnsonii X X X X L. kefiranofaciens X X X L. paracasei X X L.plantarum X X X X X L. reuteri X X X X L. rhamnosus X X X X L. ruminus XX X L. sakei X X X X X L. salivarius X X X X B. adolescentis X X X X XB. animalis X X X X X B. asteroides X X X B. bifidum X X X X X B. breveX X X X X B. dentium X X X X B. longum X X X X X B. thermophilum X X X

To test this we isolated a Lactobacillus reuteri strain from pig fecesthat is able to grow on gluconate. This strain was grown to high celldensities and 10¹⁰ CFU was used to gavage 14d old piglets daily forthree days in a pilot experiment. Fecal samples were collected prior togavage and two days thereafter, and these were analyzed by 16S rRNAamplicon sequencing. Importantly we found that relative populations ofEnterobacteriaceae decreased significantly, compared to baseline samples(FIG. 7), despite these populations remaining otherwise stable duringnursing in previous studies in age-matched pigs (FIG. 2). Thus, ourpreliminary data provides tantalizing evidence that our hypothesizedmechanism (and solution) may be effective in reducing Proteobacteriapopulations. Specifically, FIG. 7 shows the treating 14d old pigs bygavage with Lactobacillus UCD14261 led to significant reductions inEnterobacteriaceae populations.

We also identified a distinction between populations of piglets evenwithin the same litter. Some animals (7/11) harbored higher (p<0.05)populations of Enterobacteriaceae, which were, on average twice theaverage population found in low-Enterobacteriaceae animals (4/11animals) (FIG. 8). FIG. 8 gives distinct differences in “At risk” orhigh-Enterobacteriaceae versus “NR” “No risk” pigs prior to gavage withLactobacillus. These piglets responded differently to supplementedLactobacillus reuteri UCD14261, where animals harboring highEnterobacteriaceae populations (which we termed “At-Risk” (AR) animals)showed significant drops in these organisms after gavage withLactobacillus (FIG. 9), populations in the low-Enterobacteriaceaeanimals were largely unaffected. These “at risk” animals hadsignificantly lower populations of starting Lactobacillaceae populations(p<0.05), which may help explain why higher populations ofEnterobacteriaceae could thrive, and why supplementation withLactobacillus led to a reduction where populations of Enterobacteriaceaewere not significantly different from low-Enterobacteriaceae animals.FIG. 9 shows “At risk” (AR) or high-Enterobacteriaceae pigs could berescued by gavage with Lactobacillus to resemble No Risk or NonResponder animals. Letters denote significance groups (a, b; p<0.05).

FIG. 10 shows a model for PMO consumption.

Materials and Methods

All experiments involving animals were reviewed and approved by theUniversity of California Davis Institutional Animal Care and UseCommittee prior to experimentation (Approval #17776, #18279). Throughoutthe study, all animals were housed in a controlled-access specificpathogen free facility at the University of California Davis dedicatedto the rearing of pigs. Three healthy adult pregnant sows from theUniversity of California herd were selected for this study. Upondelivery, the infant pigs were cohoused with sows and ear tagged foridentification, following standard practices. The piglets were allowedto nurse freely until weaning after 21 days of age. Piglets were removedfrom the sow and transferred to separate housing and fed a standardstarter feed (Hubbard Feeds Mankato, Minn. USA) after 21 days of age.Animals were given ad libitum access to water and feed. Milk wascollected from sows while nursing their respective litters and stored at−80 C.

Sampling. Fecal samples were collected using a sterile cotton swab(Puritan Medical, Guilford, Me. USA) rectally from each piglet after 1,3, 5, 7, 14, 21, 28, 35, and 42 days after birth. Swabs were also usedto collect fecal samples from mother sows and −4 cm² sites within theenclosure throughout the study.

Sequencing Library Construction. DNA was extracted from swabs using theZymo Research Fecal DNA kit (Zymo Research Irvine, Calif. USA) accordingto the manufacturer's instructions. Extracted DNA was used as a templatefor PCR using barcoded primers to amplify the V4 region of the 16S rRNAgene as previously described for bacteria and the internal transcribedspacer region (ITS) to assess fungal communities {Bokulich:2013uf,Bokulich:2013vd}.

Briefly, the V4 domain of the 16S rRNA gene was amplified using primersF515 (5′-NNNNNNNNGTGTGCCAGCMGCCGCGGTAA-3′; SEQ ID NO:1) and R806(5′-GGACTACHVGGGTWTCTAAT-3′, SEQ ID NO. 2), where the poly-N(italicized) sequence was an 8-nt barcode unique to each sample and a2-nt linker sequence (bold). PCR amplification was carried out in a 15μL reaction containing 1× GoTaq Green Mastermix (Promega, Madison, Wis.USA), 1 mM MgCl₂ and 2 μmol of each primer. The amplification conditionsincluded an initial denaturation step of 2 minutes at 94° C., followedby 25 cycles of 94° C. for 45 seconds, 50° C. for 60 seconds, and 72° C.for 90 seconds, followed by a single final extension step at 72° C. for10 minutes. All primers used in this study are summarized in Table S1.Amplicons were pooled and purified using a Qiagen PCR purificationcolumn (Qiagen) and submitted to the JC Davis Genome Center DNATechnologies Sequencing Core for paired-end library preparation, clustergeneration and 250 bp paired-end sequencing on an Illumina MiSeq. Fungaland bacterial amplicons were sequenced in separate MiSeqrunsQuality-filtered demultiplexed reads were analyzed using QIIME 1.8.0{Kuczynski:2012gz, Walters:2010ts} as previously described{Bokulich:2014tx}, except the 13_8 greengenes database release was usedfor OTU picking and taxonomy assignment and bacterial sequences werealigned using UCLUST (Edgar:2010cv). 7 000 sequences per sample wererandomly subsampled for analysis of bacterial communities to ensuresuitable comparisons. Samples with fewer than 7 000 sequences wereomitted. Alpha diversity estimates were computed for phylogeneticdiversity (PD) whole tree and compared by nonparametric two-samplet-test with Bonferroni correction and 999 Monte Carlo permutations forbacterial analyses. Beta diversity was calculated by weighted (orunweighted, where noted) UNIFRAC metrics for bacterial populations{Lozupone:2005gn}.

Metagenome sequencing. Total genomic DNA was extracted from fecalsamples with the ZYMO Research Fecal DNA Extraction kit according tomanufacturer instructions and prepared using the Illumina MiSeq v3Reagent Chemistry for whole genome shotgun sequencing of multiplexed 150bp libraries at the University of California Davis Genomic SequencingCore (http://dnatech.genomecenter.ucdavis.edu). Samples were pooled andsequenced across triplicate sequencing runs. FASTQ files weredemultiplexed, quality filtered, trimmed to 150 bp, and then reads foreach sample were pooled from the three runs, yielding 15-20 millionreads per sample, and submitted to the MGRAST pipeline for analysis{Glass:2010ke}, which removes host genomic DNA reads and duplicatereads, bins 16S rRNA reads, and functionally classifies remaining readsby predicted protein sequence. Classified reads were normalized inMGRAST and compared between treatments using STAMP {Parks:2014hi}.

Isolation of PMG-Consuming Bacteroides and Escherichia coli. Fecalsamples were diluted in phosphate buffered saline (pH 7.0) and platedonto pre-reduced Bacteroides Bile Esculin Agar (HiMedia Mumbai, India)plates and incubated at 37° C. anaerobically for 2 d, then subculturedto purity and typed using a MALDI-TOF Biotyper (Bruker CorporationFremont Calif., USA) according to manufacturer's instructions. 16S rRNAsequencing using primers 8F and 1391R were used to confirm identity.Bacteroides were cultured in BHI-S overnight, anerobically at 37° C.Bacteroides was grown in minimal medium for growth assays, as describedpreviously {Martens:2008kp}, using lactose, glucose, galactose,2,3-sialyllactose, 2,6-sialyllactose, sialic acid as sole carbon sources(1% w/v).

Identification of sialic-acid consuming Lactobacillus species. Fecalsamples from nursing and weaned pigs were cultured on Rogosa SL mediacontaining glucose, raffinose, or ribose as sole carbon sources andgrown at 37 or 45 C anaerobically, to preferentially isolate species ofLactobacillus. Colonies were isolated to purity and initially identifiedusing a MALDI-TOF Mass Spectrometer and BioTyper system (Bruker,Fremont, Calif. USA). Genomic DNA was extracted as described previously{Oh:2009bo} and partial 16S rRNA sequences were generated by PCR usingprimers 8F and 581R under cycling and reaction conditions describedelsewhere {Bokulich:2013vd}. Isolates were grouped at the species leveland representatives selected for growth screening and 16S rRNAdetermination. Sequences were determined by the UC Davis DNA SequencingCore (http://dnaseq.ucdaysis.edu) and compared to the NCBI 16S rRNAdatabase to confirm MALDI-BioTyper identification. Representativeisolates were screened for the ability to grow on (1% w/v) sialic acidor N-acetylglucosamine as sole carbon sources in basal MRS mediumcontaining these as a sole carbon source. Lactose and glucose were alsocompared as positive controls. Lactobacillus genomes available in theJGI-IMG database were screened for the presence of a complete sialicacid utilization repertoire.

Genome Sequencing. Lactobacilli, Bacteroides spp. isolated from nursingpiglet fecal samples and possessing the sialidase predicted bymetagenomic sequencing, and the Escherichia coli containing the sialicacid catabolism pathway as determined by PCR, were selected for wholegenome shotgun sequencing on an Illumina HiSeq at the UC BerkeleyVincent J. Coates Genomics Sequencing Laboratory(http://qb3.berkeley.edu/qb3/gsl/index.cfm). Reads were assembled usingvelvet {Zerbino:2008vu}, yielding an average coverage >20-fold, anduploaded to the JGI database for annotation and public deposition.

Detection of sialic acid in feces. Fecal samples were suspended in 500uL of dH2O and vortexted for 30 m at 2500 RPM and then centrifuged at 14000 RPM for 15 minutes, from which the supernatant was removed. Twoadditional extractions of the pellet were performed for a final volumeof 1.5 mL. 150 uL was removed for protein quantification using theBradford Assay, with BSA to generate a standard curve. Samples werepurified on an anion-exchange resin and eluted with 5 mL 50 mM NaCl anddried under vacuum before reconstituting in 500 uL dH2O. Sialic acidconcentrations were determined using a commercial kit according to themanufacturer's instructions (Abcam Cambridge, Mass. USA). The sialicacid concentration is normalized to total protein concentration andexpressed as mg sialic acid per mg protein.

Statistical Analysis. T-tests and linear correlations were calculatedusing Graph Pad Prism 6 for OSX (Graph Pad Software, La Jolla, Calif.USA) with a minimum p value of 0.05.

Although the description herein contains many details, these should notbe construed as limiting the scope of the disclosure but as merelyproviding illustrations of some of the presently preferred embodiments.Therefore, it will be appreciated that the scope of the disclosure fullyencompasses other embodiments which may become obvious to those skilledin the art.

In the claims, reference to an element in the singular is not intendedto mean “one and only one” unless explicitly so stated, but rather “oneor more.” All structural, chemical, and functional equivalents to theelements of the disclosed embodiments that are known to those ofordinary skill in the art are expressly incorporated herein by referenceand are intended to be encompassed by the present claims. Furthermore,no element, component, or method step in the present disclosure isintended to be dedicated to the public regardless of whether theelement, component, or method step is explicitly recited in the claims.No claim element herein is to be construed as a “means plus function”element unless the element is expressly recited using the phrase “meansfor”. No claim element herein is to be construed as a “step plusfunction” element unless the element is expressly recited using thephrase “step for”.

In addition to any other claims, the applicant(s)/inventor(s) claim eachand every embodiment of the technology described herein, as well as anyaspect, component, or element of any embodiment described herein, andany combination of aspects, components or elements of any embodimentdescribed herein.

All cited references are incorporated herein by reference in theirentirety.

What is claimed is:
 1. A method of altering the composition of the gutbacterial microbiome in nursing non-human mammal, the method comprising:administering to a nursing non-human mammal a dose of an agentcomprising Bifidobacterium longum subsp. infantis, wherein the non-humanmammal is a horse, thereby altering the composition of the gut bacterialmicrobiome in the horse.
 2. The method of claim 1, wherein said agent isadministered at dose of 10⁴ to 10¹² colony forming units (CFU).
 3. Themethod of claim 1, further comprising administering at least oneprebiotic agent that stimulates colonization of the microorganism in themammal's gut.
 4. The method of claim 1, wherein said milk glycanmonomers further comprise at least one of the milk glycan monomersselected from the group consisting of gluconate, sialic acid, fucose,N-acetylglucosamine, N-acetylgalactosamine, galactose, glucose,N-acetylmannosamine, N-acetylmannosamine-6-Phosphate,fuculose-1-phosphate, lactaldehyde, and 1,2-propanediol,galactose-1-phosphate, and galactitol.
 5. The method of claim 1, whereinsaid agent is administered orally.
 6. The method of claim 1, whereinsaid agent further comprises a prebiotic glycan substrate and whereinsaid prebiotic glycan substrate is utilized by said bacterium.
 7. Themethod of claim 1, wherein said agent is administered orally in alyophilized form.
 8. The method of claim 1, wherein the mammal istreated with antibiotics.
 9. The method of claim 1, wherein the mammalis not treated with antibiotics.
 10. The method of claim 1, wherein themammal has diarrhea.
 11. The method of claim 1, wherein the mammal doesnot have diarrhea.
 12. The method of claim 1, wherein the mammal has anelevated gut concentration of milk glycan monomers or proteobacteriaprior to the administering.
 13. The method of claim 12, wherein thelevel of sialic acid in the mammal gut as measured in the mammal's fecesis at least 1 mg per mg total protein or the level of fucose is at least1 mg/g feces, or the level of N-acetylglucosamine orN-acetylgalactosamine is at least 10 micrograms/g feces prior to theadministering.
 14. The method of claim 12, wherein (i) at least 10% ofthe total bacteria community in the mammal's gut is Enterobacteriaceaeor Bacteroidaceae prior to the administering, or (ii) at least 10¹⁰cells per gram gut organ contents are Enterobacteriaceae orBacteroidaceae prior to the administering.
 15. The method of claim 12,wherein the mammal is tested for the presence of elevated levels of milkglycan monomers or proteobacteria prior or after the administering. 16.The method of claim 1, wherein the dose limits the availability ofgluconate in the nursing mammal's gut.
 17. The method of claim 1,wherein the mammal has an elevated gut concentration of Clostridiaceaeprior to the administering.